This invention pertains to compositions and methods for specifically inhibiting cells that are driven by or are dependent on specific ligand interactions. Examples are compositions and methods for long-term contraception or sterilization; compositions and methods for inhibiting or killing malignant and non-malignant, hormone-dependent tumors; compositions and methods for selectively killing virally infected cells; and compositions and methods for selectively destroying lymphocytes responsible for autoimmune disorders.
Compositions that have sometimes been used for long-term contraception include those based upon natural or synthetic steroidal hormones to xe2x80x9ctrickxe2x80x9d the female reproductive tract into a xe2x80x9cfalse pregnancy.xe2x80x9d These steroidal hormones must be administered repeatedly to prevent completion of the estrous cycle and conception. Steroids have side effects that can be potentially dangerous.
P. Olson et al., xe2x80x9cEndocrine Regulation of the Corpus Luteum of the Bitch as a Potential Target for Altering Fertility,xe2x80x9d J. Reprod. Fert. Suppl., vol. 39, pp. 27-40 (1989) discusses the luteal phase and its regulation in bitches. The following discussion appears at page 37: xe2x80x9cSpecific toxins can be linked to an antibody or hormone and carried to a specific target cell (or cells) which is then killed by the toxin. The idea of developing a xe2x80x98magic bulletxe2x80x99 has been discussed for decades but is now gaining renewed recognition as a potential, highly selective method for destroying specific tissues while leaving other tissues unharmed. For many years it was impossible to develop large quantities of antibodies which would react specifically with only single antigenic determinants. However, with the advent of monoclonal antibodies, this problem has been largely overcome. Antibodies can be developed to specific hormone receptors (such as the LH receptor) and then coupled to a toxin. All cells with LH receptors should then be destroyed. Although various cell types have not been characterized in dog corpora lutea, destruction of any luteal cell type could potentially result in luteolysis if cell types communicate.xe2x80x9d (citations omitted)
P. Olson et al., xe2x80x9cNew Developments in Small Animal Population Control,xe2x80x9d JAVMA, vol. 202, pp. 904-909 (1993) gives an overview of methods for preventing or terminating unwanted pregnancies in small animals. The following discussion appears at page 905: xe2x80x9cTissue-specific cytotoxinsxe2x80x94Permanent contraception in females and males might be achieved by administration of a cytotoxin that is linked to gonadotropin-releasing hormone (GnRH) and that selectively destroys gonadotropin-secreting pituitary cells. Similarly, a cytotoxin linked to antibodies against gonadotropin receptors could be targeted to alter gonadal function. Toxins would need to be carefully targeted to specific cells, yet be safe for all other body tissues.xe2x80x9d (citation omitted).
T. Janaky et al., xe2x80x9cShort Chain Analogs of Luteinizing Hormone-Releasing Hormone Containing Cytotoxic Moieties,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 89, pp. 10203-10207 (1992) discloses the use of certain hexapeptide and heptapeptide analogs of GnRH as carriers for certain alkylating nitrogen mustards, certain anthraquinone derivatives, antimetabolite, and cisplatin-like platinum complex. The authors reported that several of the compounds exerted some cytotoxic effects on the MCF-7 breast cancer cell line.
D. Fitzgerald et al., xe2x80x9cTargeted Toxin Therapy for the Treatment of Cancer,xe2x80x9d J. Natl. Cancer Inst., vol. 81, pp. 1455-1463 (1989), reviewed targeted toxin therapies for cancers, including conjugating toxins such as Pseudomonas exotoxin, diphtheria toxin, and ricin to a cell-binding protein such as a monoclonal antibody or a growth factor. The conjugates are then internalized into cytoplasm, where the toxin disrupts cellular activity.
Conventional targeted toxin therapies have several drawbacks. There is a small window for treatment with a particular targeted toxin (on the order of two weeks) before the recipient""s immune system mounts an antibody response to the targeted toxin. These antibodies will neutralize the toxin; or worse, may result in deposition of the toxin in reticuloendothelial tissues (e.g., liver, spleen, lymph nodes, lungs, bone marrow), where they may damage otherwise healthy tissue. Aside from this drawback, the toxin must be internalized by the targeted cell and translocated into the cytoplasm to have effect.
A related approach is to link a monoclonal antibody to an enzyme. This conjugate is directed specifically to a tumor cell surface antigen. A prodrug is then administered to the patient. The prodrug is substantially less toxic than the drug that results from activation of the drug at the tumor site by the conjugated enzyme. The activated drug then erectively attacks tumor cells. See, e.g., D. Kerr et al., xe2x80x9cRegressions and Cures of Melanoma Xenografts following Treatment with Monoclonal Antibody xcex2-Lactamase Conjugates in Combination with Anticancer Prodrugs,xe2x80x9d Cancer Research, vol. 55, pp. 3558-3563 (1995); and H. Svensson et al., xe2x80x9cIn Vitro and In Vivo Activities of a Doxorubicin Prodrug in Combination with Monoclonal Antibody xcex2-Lactamase Conjugates,xe2x80x9d Cancer Research, vol. 55, pp. 2357-2365 (1995).
S. Sealfon et al., xe2x80x9cMolecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor,xe2x80x9d Endocrine Reviews, vol. 18, pp. 180-205 (1997) provides a review of research concerning the interaction between GnRH and its receptor.
F. Hu et al., xe2x80x9cTheophylline and Melanocyte-Stimulating Hormone Effects on Gamma-Glutamyl Transpeptidase and DOPA Reactions in Cultured Melanoma Cells,xe2x80x9d J. Investigative Dermatology, vol. 79, pp. 57-61 (1982) disclosed that theophylline and melanocyte-stimulating hormone (MSH) both enhanced pigmentation in murine melanoma cells, apparently by different mechanisms. J. Murphy et al., xe2x80x9cGenetic Construction, Expression, and Melanoma-Selective Cytotoxicity of a Diphtheria Toxin-Related xcex1-Melanocyte-Stimulating Hormone Fusion Peptide,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 83, pp. 8258-8262 (1986) discloses selective activity against melanoma cells in vitro by an MSH-diphtheria toxin conjugate. See also D. Bard, xe2x80x9cAn Improved Imaging Agent for Malignant Melanoma, Based on [Nle4, D-Phe7]xcex1-Melanocyte Stimulating Hormone,xe2x80x9d Nucl. Med. Comm., vol. 16, pp. 860-866 (1995).
W. Siegrist et al., xe2x80x9cHomologous and Heterologous Regulation of xcex1-Melanocyte-Stimulating Hormone Receptors in Human and Mouse Melanoma Cell Lines,xe2x80x9d Cancer Research, vol. 54, pp. 2604-2610 (1994) reports that it is well-established that human melanoma cells possess specific high affinity receptors for xcex1-MSH. See also J. Tatro et al., xe2x80x9cMelanotropin Receptors Demonstrated In Situ in Human Melanoma,xe2x80x9d J. Clin. Invest., vol. 85, pp. 1825-1832 (1990).
P. Bacha et al., xe2x80x9cThyrotropin-Releasing Hormone-Diphtheria Toxin-related Polypeptide Conjugates,xe2x80x9d J. Biol. Chem., vol. 258, pp. 1565-1570 (1983) discloses conjugates of thyrotropin-releasing hormone (TRH) with two diphtheria toxins; one of these conjugates caused a 50% inhibition of protein synthesis in rat GH3 pituitary cells at 3xc3x9710xe2x88x929 M concentration. See also P. Bacha et al., xe2x80x9cOrgan-Specific Binding of a Thyrotropin-Releasing Hormone-Diphtheria Toxin Complex after Intravenous Administration to Rats,xe2x80x9d Endocrinology, vol. 113, pp. 1072-1076 (1983).
V. Chaudhary, xe2x80x9cActivity of a Recombinant Fusion Protein between Transforming Growth Factor Type xcex1 and Pseudomonas toxin,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 84, pp. 4538-4542 (1987) discloses that a fusion protein of a modified Pseudomotias toxin and transforming growth factor type xcex1 selectively kills cells expressing epidermal growth factor receptors. See also D. Cawley et al., xe2x80x9cEpidermal Growth Factor-Toxin A Chain Conjugates: EGF-Ricin 1s a Potent Toxin while EGF-Diphtheria Fragment A is Nontoxic,xe2x80x9d Cell, vol. 22, pp. 563-570 (1980).
E. Viterta et al., xe2x80x9cRedesigning Nature""s Poisons to Create Anti-Tumor Reagents,xe2x80x9d Science, vol. 238, pp. 1098-1104 (1987) reviews the use of immunotoxins against tumors. Uses in preventing graft-versus-host reactions are also mentioned. The authors mentioned that in vivo effectiveness was less than desirable. Difficulties mentioned included accessibility of toxins in circulation to target cells; instability of the linkage of toxin to antibody; rapid clearance of the immunotoxins from circulation by the liver; response by the recipient""s immune system to the toxin or to the monoclonal antibody, complicating long-term therapy; possible lack of specificity for neoplastic renewal cells; cross-reactivity with normal cells; heterogeneity of tumor cells; and shedding of surface antigens by tumor cells.
P. Trail et al., xe2x80x9cAntigen-specific Activity of Carcinoma-reactive BR64-Doxorubicin Conjugates Evaluated in Vitro and in Human Tumor Xenograft Models.xe2x80x9d Cancer Research, vol. 52, pp. 5693-5700 (1992) disclose the conjugation of the anticarcinoma antibody BR64 to a doxorubicin derivative, and discuss the antitumor effects of the conjugate.
J. Olson, xe2x80x9cLaboratory Evidence for the Hormonal Dependency of Meningiomas,xe2x80x9d Human Reproduction, vol. 9, supp. 1, pp. 195-201 (1994) discloses evidence that meningiomas, benign intracranial tumors, possess progesterone receptors.
S. Prigent et al., xe2x80x9cThe Type 1 (EGFR-Related) Family of Growth Factor Receptors and their Ligands,xe2x80x9d Progress in Growth Factor Research, vol. 4, pp. 1-24 (1992) reviews the biology of the epidermal growth factor (EGF), its receptor, and related ligands and receptors (e.g., c-erbB-2, c-erbB-3, TGFxcex1, amphiregulin, heregulin), and their roles in normal cell proliferation and in the pathogenesis of human cancer. See also D. Davies et al., xe2x80x9cTargeting the Epidermal Growth Factor Receptor for Therapy of Carcinomas,xe2x80x9d Biochem. Pharm., vol. 51, pp. 1101-1110 (1996).
D. Morbeck et al., xe2x80x9cA Receptor Binding Site Identified in the Region 81-95 of the xcex2-Subunit of Human Luteinizing Hormone (LH) and chorionic gonadotropin (hCG),xe2x80x9d Molecular and Cellular Endocrinology, vol. 97, pp. 173-181 (1993) disclosed a fifteen amino acid region of LH and hCG that acted as a receptor binding site. (LH and hCG are homologous hormones that produce similar effects.)
W. Theunis et al., xe2x80x9cLuteinising Hormone, Follicle Stimulating Hormone and Gonadotropin Releasing Hormone Immunoreactivity in Two Insects: Locusia migratoria migratoroides R and F and Sarcophaga bullata (Parker),xe2x80x9d Invert. Reprod. and Develop., vol. 16, pp. 111-117 (1989) disclosed that materials immunologically related to LH, FSH, and GnRH were localized in cerebral tissue of Locusia migraforia and Sarcophaga bullafa. See also P. Verhaert et al., xe2x80x9cSubstances Resembling Peptides of the Vertebrate Gonadotropin System Occur in the Central Nervous System of Periplaneta americana L.,xe2x80x9d Insect Biochem., vol. 16. pp. 191-197 (1986).
U.S. Pat. Nos. 5,378,688; 5,488,036; and 5,492,893 disclose compounds said to be useful in inducing sterility in mammals, and in treating certain sex hormone-related cancers in mammals. The disclosed compounds were generically described as GnRH (or a GnRH analog) conjugated to a toxin. The toxin was preferably linked to the sixth amino acid of the GnRH agonist. The toxin was preferably one with a translocation domain to facilitate uptake into a cell. The inventors noted that conjugation of the GnRH agonist to the toxin xe2x80x9cis necessary because, for the most part, the above toxins, by themselves, are not capable of binding with cell membranes in general. That is to say that applicants have found that it is only when a GnRH analog of the type described herein is linked to a toxin of the type noted above does that toxin become capable of binding to cell membranes . . .xe2x80x9d (E.g., U.S. Pat. No. 5,488,036, col. 7, lines 46-52.) The toxins specifically mentioned appear all to have been metabolic toxins, for example ricin, abrin, modeccin, various plant-derived ribosome-inhibiting proteins, pokeweed antiviral protein, xcex1-amanitin, diphtheria toxin, pseudomonas exotoxin, shiga toxin, melphalan, methotrexate, nitrogen mustard, doxorubicin, and daunomycin. None of these toxins is believed to be toxic due to direct interaction with the cell membrane. In the in vivo experiments reported, the most effective time course was reported to be weekly injections for 4 weeks. (E.g., U.S. Pat. No. 5,488,036, col. 20, lines 46-47.) Because most of the conjugates cited are relatively large compounds, antigenicity could be a problem when such multiple administrations are used. The GnRH analog was preferably linked to the toxin with one of several specified heterobifunctional reagents. The specifications suggest that considerable effort was expended in conjugating the toxin to the GnRH agonist. The toxins must in general be internalized into the target cells to have effect, and do not act on cell membranes; in addition, at least some of these toxins must be secondarily transported from the membrane-bound vesicle into the cytoplasm to interact with ribosomes, mitochondria, or other cellular components.
M. Kovacs et al., xe2x80x9cRecovery of pituitary function after treatment with a targeted cytotoxic analog of luteinizing hormone-releasing hormone,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 94, pp. 1420-1425 (1997) discloses that a doxorubin analog conjugated to an LH-RH (i.e., GnRH) agonist selectively attacked cells with LH-RH receptors, and that its effect on pituitary cells was reversible. The paper suggests that the conjugate might be used to treat tumors with LH-RH receptors. See also A. Jungwirth et al., xe2x80x9cRegression of rat Dunning R-3227-H prostate carcinoma by treatment with targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 containing 2-pyrrolinodoxorubicin,xe2x80x9d Intl. J. Oncol., vol. 10, pp. 877-884 (1997)
R. Moretti et al., xe2x80x9cLuteinizing hormone-releasing hormone agonists interfere with the stimulatory actions of epidermal growth factor in human prostatic cancer cell lines, LNCaP and DU 145,xe2x80x9d J. Clin. Endocrin. and Metab., vol. 81, pp. 3930-3937 (1996) discloses that LH-releasing hormone agonists inhibit both androgen-dependent (LNCaP) and androgen-independent (DU 145) human prostatic cancer cell lines, and suggests that the agonists may inhibit proliferation of the tumor cells by interfering with the stimulatory actions of epidermal growth factor.
I. Mezxc3x4 et al., xe2x80x9cSynthesis of GnRH analogs having direct antitumor and low LH-releasing activity,xe2x80x9d J. Med. Chem., vol. 40, pp. 3353-3358 (1997) discloses chicken 1 GnRH agonists and antagonists. Agonist MI-1892 was reported to have low endocrinological activity, but to possess antitumor activity.
A. Nechushtan et al., xe2x80x9cAdenocarcinoma cells are targeted by the new GnRH-PE66 chimeric toxin through specific gonadotropin-releasing hormone binding sites,xe2x80x9d J. Biol. Chem., vol. 272, pp. 11597-11603 (1997) discloses the use of a Pseudonionas exotoxin coupled to GnRH to kill certain types of cancer cells.
X. Zhu, xe2x80x9cSteroid-independent activation of androgen receptor in androgen-independent prostate cancer. A possible role for the MAP kinase signal transduction pathway?xe2x80x9d Mol. and Cell. Endocrinol., vol. 134, pp. 9-14 (1997) discloses that androgen receptors in prostate cancer could be activated in the absence of the androgen signal.
G. Emons et al., xe2x80x9cGrowth-inhibitory actions of analogues of luteinizing hormone releasing hormone on tumor cells,xe2x80x9d Trends in Endocrin. Metab., vol. 8, pp. 355-362 (1997) reviews the similarities and differences between GnRH receptors of cancer cells and of normal brain and pituitary cells; and suggests that LHRH analogs interfere with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity in a number of malignant human tumors, including breast, ovary, endometrium, and prostate cancers.
D. Tang et al., xe2x80x9cTarget to Apoptosis: A Hopeful Weapon for Prostate Cancer,xe2x80x9d The Prostate, vol. 32, pp. 284-293 (1997) provides a review of research on apoptosis as a route to treat prostate cancers.
A. Goustin et al., xe2x80x9cGrowth Factors and Cancer,xe2x80x9d Cancer Research, vol. 46, pp. 1015-1029 (1986) provides an overview of various growth factors that have been associated with different cancers.
S. Cho et al., xe2x80x9cEvidence for autocrine inhibition of gonadotropin-releasing hormone (GnRH) gene transcription by GnRH in hypothalamic GT1-1 neuronal cells,xe2x80x9d Mol. Brain Res., vol. 50, pp. 51-58 (1997) discloses that neuroendocrine populations of GnRH neurons have high affinity receptors for GnRH and for GnRH analogs.
S. Sower et al., xe2x80x9cPrimary structure and biological activity of a third gonadotropin-releasing hormone from lamprey brain,xe2x80x9d Endocrinology, vol. 132, pp. 1125-1131 (1993) describes the structure of lamprey III GnRH.
E. Stopa et al., xe2x80x9cImmunocytochemical evidence for a lamprey-like gonadotropin-releasing hormone in human brain,xe2x80x9d Soc. Neurosci. Abstr., abstract no. 437.8, p. 1577 (1987) discloses that a lamprey-like GnRH III is found in humans.
S. White et al., xe2x80x9cThree gonadotropin-releasing hormone genes in one organism suggest novel roles for an ancient peptide,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 92, pp. 8363-8367 (1995); and J. Powell et al., xe2x80x9cThree forms of gonadotropin-releasing hormone characterized from brains of one species,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 91, pp. 12081-12085 (1994) are examples of papers reporting the typical presence of three forms of GnRH in species of vertebrates.
J. Warnock et al., xe2x80x9cAnxiety and mood disorders associated with gonadotropin-releasing hormone agonist therapy,xe2x80x9d Psychopharmacology Bull., vol. 33, pp. 311-316 (1997) reports that psychological side effects can accompany chronic treatment with a GnRH agonist.
L. Deligdisch et al., xe2x80x9cPathological changes in gonadotropin releasing hormone agonist analogue treated uterine leiomyomata,xe2x80x9d Fertility and Sterility, vol. 67, pp. 837-841 reported the pathological changes associated with treating leiomyomata with a GnRH analog to induce iatrogenic menopause.
J. Fuerst et al., xe2x80x9cEffect of active immunization against luteinizing hormone-releasing hormone on the androgen-sensitive Dunning R3327-PAP and Androgen-Independent Dunning R3327-AT2.1 prostate cancer sublines,xe2x80x9d Prostate, vol. 32, pp. 77-84 (1997) reported that active immunization of rats with an LHRH-diphtheria toxoid conjugate caused atrophy of the testes, prostate, and androgen-sensitive prostate tumors, with inhibition of the tumors caused by suppression of cell division rather than an increase in cell death; and that the volume increase of androgen-independent prostate tumors was slightly reduced.
C. Mantzoros et al., xe2x80x9cInsulin-like growth factor 1 in relation to prostate cancer and benign prostatic hyperplasia,xe2x80x9d Br. J. Cancer, vol. 76, pp. 1115-1118 (1997) reported that increased levels of insulin-like growth factor 1 were associated with an increased risk of prostate cancer.
V. Ding, xe2x80x9cSex hormone-binding globulin mediates prostate androgen receptor action via a novel signaling pathway,xe2x80x9d Endocrinology, vol. 139, pp. 213-218 (1998) reported that androgen-independent pathways may activate the progression of some prostate cancers.
J. King et al., xe2x80x9cEvolution of gonadotropin-releasing hormones,xe2x80x9d Trends in Endocrin. Metab., vol. 3, pp. 339-344 (1992) discloses the primary structures of different GnRHs from various vertebrates. See also J. King et al., xe2x80x9cStructure of chicken hypothalamic luteinizing hormone-releasing hormone. II. Isolation and characterization,xe2x80x9d J. Biol. Chem., vol. 257, pp. 10729-10732 (1982).
N. Mores et al., xe2x80x9cActivation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release,xe2x80x9d Endocrinology, vol. 137. pp. 5731-5734 (1996) discloses that LH acts directly on neuroendocrine neurons in the brain. See also Z. Lei et al., xe2x80x9cSignaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons,xe2x80x9d Mol. and Cell. Endocrinology, vol. 109, pp. 151-157 (1995).
U.S. Pat. Nos. 5,597,945 and 5,597,946 disclose plants transformed with genes encoding various lytic peptides.
It has been unexpectedly discovered that amphipathic lytic peptides are ideally suited to use in a ligand/cytotoxin combination to specifically inhibit abnormal or normal cells that are driven by or are dependent upon a specific ligand interaction; for example, to induce sterility or long-term contraception, or to attack tumor cells, or to selectively lyse virally-infected cells, or to attack lymphocytes responsible for autoimmune diseases. The peptides act directly on cell membranes, and need not be internalized.
For example, administering a combination of gonadotropin-releasing hormone (GnRH) (or a GnRH agonist) and a membrane-active lytic peptide produces long-term contraception or sterilization in animals in vivo. Particularly surprising, sterility results even when the combination is administered to a sexually immature animal: The combination then prevents sexual maturation.
Administering in vivo a combination of a ligand and a membrane-active lytic peptide kills cells with a receptor for the ligand. The compounds used in the present invention are relatively small, and will not be antigenic. (Lytic peptides are known not to be very antigenic; and the ligands are not antigenic at all.) The compounds may be administered in a single dose, or in two or more closely spaced doses. Lysis of gonadotropes has been observed to be very rapid (on the order of ten minutes.) Lysis of tumor cells is rapid. The two componentsxe2x80x94the ligand and the lytic peptidexe2x80x94may optionally be administered as a fusion peptide, or they may be administered separately, with the ligand administered slightly before the lytic peptide, to activate cells with receptors for the ligand, and thereby make those cells susceptible to lysis by the lytic peptide. If a fusion peptide is used, it has been unexpectedly discovered that a linking moiety is not necessary to join the ligand to the lytic peptide: one may be bonded directly to the other, without the need for any intervening linkage; bonding may be performed by bonding one end of the ligand to one end of the peptide, or by bonding to the middle of either. The toxin, the lytic peptide, does not need a translocation domain, and need not be internalized, as it binds to and acts directly on the activated cell membrane to cause lysis. The ligand may be a full native compound, or it may instead be the binding domain alone; the latter is preferred where the full ligand is relatively large.
The compounds of the present invention are well-suited for use in gene therapy to treat malignant or non-malignant tumors, and other diseases caused by clones or populations of xe2x80x9cnormalxe2x80x9d host cells bearing specific receptors (such as lymphocytes), because genes encoding a lytic peptide or encoding a lytic peptide/peptide hormone fusion may readily be inserted into hematopoietic stem cells or myeloid precursor cells.
Several cancer cells (uterine, endometrial, prostate, testicular, and ovarian) express LH or hCG receptors. Tao et al., xe2x80x9cExpression of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Gene in Benign Prostatic Hyperplasia and in Prostatic Carcinoma in Humans,xe2x80x9d Biol. Reprod., vol. 56, pp. 67-72 (1997). Conjugates of a lytic peptide and LH or a portion of the LH molecule may thus be used to destroy these cells selectively. For example, the genes encoding such hormones as FSH, TRH, and LH are known, and may be linked to a DNA sequence encoding a lytic peptide to produce a secreted fusion peptide, all under the control of a suitable promoter such as the acute-phase responsive promoters disclosed in United States patent application Ser. No. 08/474,678, filed Jun. 7, 1995, and in PCT application WO 95/01095, published Jan. 12, 1995. A binding site from a hormone may be used in lieu of the entire hormone, for example the fifteen amino acid binding site of LH and hCG. See D. Morbeck et al., xe2x80x9cA Receptor Binding Site Identified in the Region 81-95 of the xcex2-Subunit of Human Luteinizing Hormone (LH) and chorionic gonadotropin (hCG),xe2x80x9d Molecular and Cellular Endocrinology, vol. 97, pp. 173-181 (1993).
A powerful vector that is suitable for transforming cells to be used in gene therapy is the transposon-based vector that is disclosed in U.S. Pat. No. 5,719,055.
It is known that the D-amino acid form of GnRH will bind to gonadotropes in the pituitary, to GnRH neurons in the brain, and to various types of cancer cells. It is also known that the D-amino acid forms of lytic peptides have essentially the same propensity to lyse cell membranes as do the L-amino acid forms. Compounds of the present invention (whether administered as a fusion peptide or separately) may therefore be administered either in L-form or D-form. D-form peptides, although generally more expensive than L-form, have the advantage that they are not degraded by normal enzymatic processes, so that the D-form peptides may therefore be administered orally and generally have a longer biological half-life. Oral administration of the D-form peptide may be enhanced by linking the peptide/hormone fusion product to a suitable carrier to facilitate uptake by the intestine, for example vitamin B12, following generally the B12-conjugation technique of G. Russell-Jones et al., xe2x80x9cSynthesis of LHRH Antagonists Suitable for Oral Administration via the Vitamin B12 Uptake System,xe2x80x9d Bioconjugate Chem., vol. 6, pp. 3442 (1995).
GnRH or GnRH analogs (collectively, xe2x80x9cGnRH agonistsxe2x80x9d) may be used in the present invention. It has been reported that substitutions at the 6 and 10 positions of the GnRH decapeptide can produce xe2x80x9csuperagonistsxe2x80x9d having greater binding affinity to the GnRH receptor than does GnRH itself. These xe2x80x9csuperagonistsxe2x80x9d include goserelin, leuprolide, buserelin, and nafarelin. See U.S. Pat. No. 5,488,036.
Without wishing to be bound by this theory, it is believed that a mechanism (though not the exclusive mechanism) underlying the sterilization/long term contraception aspect of this invention is as follows: GnRH activates gonadotropic cells in the pituitary gland, as well as neuroendocrine GnRH neurons in the brain. The activated cells have substantially increased susceptibility to lysis by a lytic peptide. The lytic peptide then preferentially destroys (or severely damages) these activated cells. When the gonadotrophic cells in the pituitary are destroyed and are deprived of GnRH from the brain, the pituitary no longer secretes follicle stimulating hormone (FSH) or luteinizing hormone (LH), rendering the animal temporarily or permanently sterile.
Although the ligand and the lytic peptide may be administered separately, it is preferred to link the two in a single molecule, because such a linkage greatly increases the effective concentration of the lytic peptide in the vicinity of ligand-activated cells. Furthermore, this increase in the effective lytic peptide concentration can obviate the need for activation of the cells, allowing the peptide to be linked to a binding site of a ligand alone, without needing to include the xe2x80x9cremainderxe2x80x9d of a native ligand that would normally be needed for activating the target cells. This linkage may be in either order: for example, GnRH/peptide or peptide/GnRH. Examples are modified GnRH/hecate (SEQ. ID NO. 3) and hecate/modified GnRH (SEQ. ID NO. 4). Note that no intermediate linker is necessary, and that the carboxy terminus of one of the two peptides may be bonded directly to the amino terminus of the other. (We have found that the initial pyro-glutamic acid residue of GnRH or of the GnRH portion of a fusion peptide may be substituted with glutamine without substantially changing the activity of the respective peptides. See, e.g., SEQ. ID Nos. 9, 3, and 4.)